qHTS Assay for Identification of Small Molecule Inducers of Hypoxia Response

Hypoxia-inducible factor-1 (HIF-1) is the major hypoxia-regulated transcription factor that controls cellular responses to low oxygen concentration. HIF-1 is composed of two subunits: hypoxia responsive HIF-1a and constitutively expressed HIF-1b, which is also known as aryl hydrocarbon receptor nuclear translocator. During hypoxic conditions, HIF-1a heterodimerizes with HIF-1b and translocates more ..

Hypoxia-inducible factor-1 (HIF-1) is the major hypoxia-regulated transcription factor that controls cellular responses to low oxygen concentration. HIF-1 is composed of two subunits: hypoxia responsive HIF-1a and constitutively expressed HIF-1b, which is also known as aryl hydrocarbon receptor nuclear translocator. During hypoxic conditions, HIF-1a heterodimerizes with HIF-1b and translocates into the nucleus where the HIF-1 complex binds to the hypoxia-response element (HRE) and activates expression of target genes such as VEGF (vascular endothelial growth factor). The HIF-1 pathway is essential to normal growth and development, and is involved in the pathophysiology of cancer, inflammation, and ischemia.

Using a beta-lactamase reporter-gene under control of the hypoxia response element, a cell-based assay [CellSensor(TM) HRE-bla ME-180 developed by Invitrogen Corp.] was used to measure hypoxia-induced signaling. Library compounds were measured for their ability to induce the reporter gene activity. Compounds were screened in a titration series in 1536-well format. The inducers were identified based on the AC50 values derived from the screening data.

2500 cells/5uL in Opti-MEM medium containing 0.5% dialyzed FBS, 0.1 mM NEAA, 1 mM sodium pyruvate and 10 mM HEPES was dispensed into 1536-well plates and cells were cultured at 37 degrees Celsius for 4-5 hours and then 23 nL of compounds or DMSO were delivered to each well using a pin tool. The plates were incubated at 37 degrees Celsius for 17 hours. 1 uL of CCF4-dye mixture was added to each well. After incubated at room temperature for 2.5 hours the plates were measured on an EnVision plate reader at Excitation 405nm, Emission1=460nm and Emission 2=530nm. The %Activity was determined from the ratio of 460nm/530nm.

1. Compounds are first classified as having full titration curves, partial modulation, partial curve (weaker actives), single point activity (at highest concentration only), or inactive. See data field "Curve Description". For this assay, apparent activators are ranked higher than compounds that showed apparent inhibition.2. For all inactive compounds, PUBCHEM_ACTIVITY_SCORE is 0. For all active compounds, a score range was given for each curve class type given above. Active compounds have PUBCHEM_ACTIVITY_SCORE between 40 and 100. Inconclusive compounds have PUBCHEM_ACTIVITY_SCORE between 1 and 39. Fit_LogAC50 was used for determining relative score and was scaled to each curve class' score range.

Concentration at which compound exhibits half-maximal efficacy, AC50. Extrapolated AC50s also include the highest efficacy observed and the concentration of compound at which it was observed.

Float

μM

3

Efficacy

Maximal efficacy of compound, reported as a percentage of control. These values are estimated based on fits of the Hill equation to the dose-response curves.

Float

%

4

Analysis Comment

Annotation/notes on a particular compound's data or its analysis.

String

5

W460-Curve_Description

A description of dose-response curve quality. A complete curve has two observed asymptotes; a partial curve may not have attained its second asymptote at the highest concentration tested. High efficacy curves exhibit efficacy greater than 80% of control. Partial efficacies are statistically significant, but below 80% of control.

String

6

W460-Fit_LogAC50

The logarithm of the AC50 from a fit of the data to the Hill equation (calculated based on Molar Units).

Efficacy at zero concentration of compound from a fit of the data to the Hill equation.

Float

%

11

W460-Fit_CurveClass

Numerical encoding of curve description for the fitted Hill equation.

Float

12

W460-Excluded_Points

Which dose-response titration points were excluded from analysis based on outlier analysis. Each number represents whether a titration point was (1) or was not (0) excluded, for the titration series going from smallest to highest compound concentrations.

A description of dose-response curve quality. A complete curve has two observed asymptotes; a partial curve may not have attained its second asymptote at the highest concentration tested. High efficacy curves exhibit efficacy greater than 80% of control. Partial efficacies are statistically significant, but below 80% of control.

String

29

Ratio-Fit_LogAC50

The logarithm of the AC50 from a fit of the data to the Hill equation (calculated based on Molar Units).

Efficacy at zero concentration of compound from a fit of the data to the Hill equation.

Float

%

34

Ratio-Fit_CurveClass

Numerical encoding of curve description for the fitted Hill equation.

Float

35

Ratio-Excluded_Points

Which dose-response titration points were excluded from analysis based on outlier analysis. Each number represents whether a titration point was (1) or was not (0) excluded, for the titration series going from smallest to highest compound concentrations.

A description of dose-response curve quality. A complete curve has two observed asymptotes; a partial curve may not have attained its second asymptote at the highest concentration tested. High efficacy curves exhibit efficacy greater than 80% of control. Partial efficacies are statistically significant, but below 80% of control.

String

58

W530-Fit_LogAC50

The logarithm of the AC50 from a fit of the data to the Hill equation (calculated based on Molar Units).

Efficacy at zero concentration of compound from a fit of the data to the Hill equation.

Float

%

63

W530-Fit_CurveClass

Numerical encoding of curve description for the fitted Hill equation.

Float

64

W530-Excluded_Points

Which dose-response titration points were excluded from analysis based on outlier analysis. Each number represents whether a titration point was (1) or was not (0) excluded, for the titration series going from smallest to highest compound concentrations.